Method for detecting microorganisms by separation and culture on a gelled system, gelled system and assay kit therefor, and use thereof in microbiology

ABSTRACT

A centrifuge tube and a kit for detecting the presence or absence of microorganisms, particularly bacteria and yeasts, in a liquid sample of a biological material. The centrifuge tube has a cylinder portion; a frustoconical portion; a capillary at the bottom of the tube; and contains a gel system for detecting microorganisms. The gel system includes a development phase of a gel containing microorganism culture medium and a reagent capable of inducing a detectable variation in optical measurement in the presence of microorganisms and a first phase intimate mixture which includes water originating wholly or partly from the culture medium and water-absorbing polymer particles so that, in the first phase intimate mixture, the polymer particles have a particular concentration and size.

This application is a division of application Ser. No. 08/913,757 filedSep. 22, 1997 now US. Pat. No. 6,020,150.

FIELD OF THE INVENTION

The present invention relates to a novel method of detecting thepresence or absence of microorganisms belonging to the group comprisingbacteria and yeasts. This method uses a technique involving separation,culture and development on one and the same gelled system.

It further relates on the one hand to said gelled system as a novelindustrial product, and on the other hand to the assay kit for carryingout said method.

Finally, it relates to the use of said method and said gelled system inmicrobiology (especially in bacteriology or mycology), particularly inthe field of the detection of bacterial infections (more particularlythe detection of urinary infections and septicemia).

PRIOR ART

When it is desired to assess the presence or absence of microorganismssuch as bacteria and yeasts, especially by a colorimetric method, thefollowing results have to be considered:

true positives (TP),

false positives (FP),

true negatives (TN) and

false negatives (FN).

In practical terms, the following four values (expressed as percentages)have been defined from the TP, FP, TN and FN results which aretheoretically possible:

the sensitivity (Sen),

the specificity (Spe),

the positive predictive value (PPV) and

the negative predictive value (NPV),

said values being such that:

Sen=100.TP/(TP+FN),

Spe=100.TN/(TN+FP),

PPV=100.TP/(TP+FP) and

NPV=100.TN/(TN+FN),

in order to evaluate the significance or the pertinence of themeasurements.

It is known on the one hand that a colorimetric detection or filtrationtechnique has already been recommended or used for assessing thepresence or absence of bacteria in urine, and on the other hand that agel separation technique has already been recommended or used forrevealing erythrocyte agglutinates.

As far as colorimetric detection is concerned, the publication EP-A-0496 409 has disclosed a method of revealing Gram-negative bacteria. Thismethod comprises the filtration of a biological sample (especiallyurine) containing bacteria on a porous support (pore diameter 0.75-1.2μm) in order to retain the bacteria on said support, and then thedetection of the Gram-negative bacteria by reaction of theirdehydrogenases with a redox color reagent (especially a tetrazolium saltor resazurin), the reduction reactions induced by the Gram-positivebacteria being inhibited by means of a caotropic agent and a non-ionicdetergent of the alkylglucoside type.

As far as the colorimetric filtration technique is concerned, testsmarketed by VITEK SYSTEMS (Hazlewood, Mo.) under the names"Bac-T-Screen®" (semi-automatic test) and "FiltraCheck®-UTI" (manualtest) are known in particular.

These tests are performed by filtering a urine sample on a filter paperimpregnated with a developer in order to retain and color the bacteriawhich may be present in the sample; the filtration is carried out undera pressure difference, so a relatively complicated apparatus isrequired; the main source of errors (FP and FN results) is due to thepossible presence of a large number of erythrocytes, leukocytes and/orepithelial cells in the urine (the presence of erythrocytes and/orleukocytes in the urine is already in itself a sign of dysfunction).

According to the literature--cf. (a) page 272 (Table 3) and page 274(section "Colorimetric Filtration") of the article by M. PEZZLO, Clin.Microbiol. Rev., 1988, 1 (No. 2), 268-280, and (b) page 86 (paragraph"Systeme coloration filtration" ("Coloration/filtration system") andTables) of the article by F. W. GOLDSTEIN, Med Mal. Infect., 1991, 21,83-88--the Bac-T-Screen® and FiltraCheck®-UTI tests are (i) rapid (1-2minutes), (ii) effective when the bacterial population is ≧10⁵ CFU/ml,but (iii) unsatisfactory when said bacterial population is below 10⁵CFU/ml. Overall, taking these two tests together, the two articles citedabove give the following results for urine screenings according to thebacterial populations:

≧10⁵ CFU/ml: Sen=93-95%, Spe=76-77%, PPV=32% and NPV=97-99%;

≧10⁴ CFU/ml: Sen=85-89%, Spe=81% and NPV=84-86%; and

≧10³ CFU/ml: Sen=76-84% and NPV=67-72%.

There is therefore a need to improve the reliability of the measurementsfor bacterial populations below 10⁵ CFU/ml.

As far as the gel separation technique for assessing erythrocyteagglutination is concerned, the publications FR-A-2 577 321 and EP-A-0454 509 are known.

According to FR-A-2 577 321, the formation or presence of erythrocyteagglutinates is revealed by (1) the deposition of a liquid mediumcontaining erythrocytes on a gel which (i) is located in aconical-bottomed centrifuge tube and (ii) allows the free corpuscles topass through more easily than the agglutinates [especially a gelmarketed under the name "Sephade® G 100 Ultrafine" by PHARMACIA FINECHEMICALS (Uppsala, Sweden)], and then (2) gentle centrifugation.

The procedure according to EP-A-0 454 509 is substantially analogous andinvolves gentle centrifugation, the gel of FR-A-2 577 321 being replacedwith a gelled system consisting of a mixture of two dextran gels (cf.especially page 3 lines 2-4 and page 4 lines 6-7 of EP-A-0 454 509),namely the product "Sephadex® G 100 Ultrafine" mentioned above, which,in the dry state, is in the form of particles with a diameter of 20 to50 μm, and the product "Sephadex® HP Ultrafine", which, in the drystate, is in the form of particles with a diameter of 13 to 23 μm.

According to the information provided in EP-A-0 454 509 (page 3 lines2-4 and 42-43 and page 4 lines 14-15), it seems clear that the gelledsystem of EP-A-0 454 509 contains only one phase.

As described in FR-A-2 577 321 and EP-A-0 454 509, the gel separationtechnique with centrifugation is not directly applicable to thedetection of the presence or absence of microorganisms. In particular,the gels recommended, namely the products "Sephadex® G 100 Ultrafine"and "Sephadex® HP Ultrafine", do not allow the passage of microorganismssuch as bacteria and yeasts during the gentle centrifugation [i.e.according to the prior art: an intensity less than or equal to 1500 g,in particular an intensity less than 1000 g (especially of the order of400-500 g, EP-A-0 454 509 indicating, on page 4 line 49, an intensity of430 g)], nor the separation of bacteria from the other cells.

OBJECT OF THE INVENTION

According to the invention, it is proposed to provide a novel technicalsolution for detecting, in a sample of a biological material, thepresence or absence of microorganisms belonging to the group comprisingbacteria and yeasts, this novel technical solution giving more reliableresults for microbial populations below 10⁵ germs/ml (hereafter, for thesake of convenience, 1 bacterium/ml or 1 yeast/ml will be considered tocorrespond approximately to 1 CFU/ml) than the above-mentioned"Bac-T-Screen®" and "FiltraCheck®-UTI" tests relating to the evaluationof bacterial urinary infections.

This novel technical solution uses a particular technique for theseparation of microorganisms on a gelled system containing a culturemedium which is different from the technique proposed by thepublications FR-A-2 577 321 and EP-A-0 454 509 cited above, and whichavoids isolation of the microorganisms on a standard agar system or thecultivation necessary for their multiplication, prior tocentrifuigation.

SUBJECT OF THE INVENTION

This object is achieved by the use of a gelled system which on the onehand selectively separates the microorganisms which may be present in asample of a biological material from the other components, especiallythe cells, the peptides and the proteins, and which on the other handallows said microorganisms to grow in situ during the centrifilgationand then, if appropriate, after said centrifugation and theirdevelopment.

According to a first feature of the invention, a novel method isrecommended for detecting the presence or absence of microorganismsbelonging to the group comprising bacteria and yeasts in a liquid sampleof a biological material which may contain cells other than those ofsaid microorganisms, said method, which uses a gel separation technique,being characterized in that it comprises the steps consisting in:

(1°) using a gelled system which (i) separates, according to their size,the microorganisms which may be present in said liquid sample, and whichoriginate from said biological material, from the other solid componentswhich may be present in said liquid sample, (ii) is essentiallyimpermeable to the water which may be present in said liquid sample andto the dissolved substances which may be present in said liquid sample,and (iii) comprises at least

(a) a first, so-called development phase which is a gel containing amicroorganism culture medium and a reagent which induces a detectablevariation in optical measurement in the presence of microorganisms, saidgel being an intimate mixture of water and water-absorbing polymerparticles which have been swollen so that, in said intimate mixture,said polymer particles have a concentration by dry weight of between0.05 and 0.2 g/ml and a diameter in the swollen state of between 90 and320 μm, the water of said intimate mixture originating wholly or partlyfrom said culture medium;

(2°) introducing said liquid sample into a centrifuge tube on top ofsaid gelled system, which has been placed in said centrifuge tubebeforehand;

(3°) centrifuging the resulting contents of said tube; and

(4°) revealing the presence or absence of microorganisms in said liquidsample originating from said biological material, in said first phase ofthe gelled system, by means of said reagent which induces a detectablevariation in optical measurement.

If the concentration of the microorganisms in said liquid sample is toolow, the development of step (4°) is carried out after multiplication ofthe microorganisms in said culture medium at a temperature between roomtemperature and 45° C., in at most 24 h (preferably in 1-2 h) in thecase of bacteria and in at most 36 h (preferably in 24-30 h) in the caseof yeasts.

According to a second feature of the invention, said gelled system which(i) separates, according to their size, the microorganisms which may bepresent in said liquid sample, and which originate from said biologicalmaterial, from the other solid components which may be present in saidliquid sample, (ii) is essentially impermeable to the water which may bepresent in said liquid sample, and (iii) comprises at least

(a) a first, so-called development phase which is a gel containing amicroorganism culture medium and a reagent which induces a detectablevariation in optical measurement in the presence of microorganisms, saidgel being an intimate mixture of water and water-absorbing polymerparticles which have been swollen so that, in said intimate mixture,said polymer particles have a concentration by dry weight of between0.05 and 0.2 g/ml and a diameter in the swollen state of between 90 and320 μm, the water of said intimate mixture originating wholly or partlyfrom said culture medium,

is recommended as a novel industrial product.

According to yet another feature of the invention, an assay kit isrecommended which comprises in particular:

said gelled system or the phases which make it up, and

if appropriate, lyophilized reference strains of microorganisms and/ortheir culture media to allow the preparation of standard samples.

Finally, according to another feature of the invention, the use of saidmethod and said gelled system is recommended in microbiology, especiallyin (i) the detection of bacterial infections (particularly such asurinary infections and septicemia), (ii) the identification of strainsof microorganisms, and (iii) the evaluation of the antibiotic resistanceof strains of microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The attached drawings schematically show centrifuge tubes each packedwith a gelled system according to the invention:

FIG. 1 shows a centrifuge tube containing a gelled system consisting ofa single phase, namely the so-called development phase;

FIGS. 2a and 2b show a centrifuge tube containing a gelled systemconsisting of two phases, namely, from bottom to top, the developmentphase and the protection phase, as it appears before centrifugation(FIG. 2a) and after centrifugation (FIG. 2b);

FIG. 3 shows a centrifuge tube containing a gelled system consisting oftwo phases, namely, from bottom to top, the development phase and theabsorption phase;

FIGS. 4a and 4b show a gelled system analogous to that of FIG. 3 whichalso contains the protection phase, as it appears before centrifugation(FIG. 4a) and after centrifugation (FIG. 4b);

FIG. 5 shows a centrifuge tube containing a gelled system consisting ofthree phases, namely, from bottom to top, the barrier phase, thedevelopment phase and the absorption phase;

FIGS. 6a and 6b show a gelled system analogous to that of FIG. 5 whichalso contains the protection phase, as it appears before centrifugation(FIG. 6a) and after centrifugation (FIG. 6b);

FIGS. 7 and 8 each show a preferred gelled system according to theinvention, in which the development phase has a small thickness;

FIG. 9 shows a cutaway of a centrifuge tube according to the invention,provided with a capillary at the bottom; and

FIG. 10 shows a front view of another centrifuge tube according to theinvention, which is also provided with a capillary.

ABBREVIATIONS

For the sake of convenience, the following abbreviations and acronymshave been used in the text of the present invention:

    ______________________________________                                        Ala   alanyl                                                                    TB tetrazolium blue                                                           CFU colony forming unit                                                       DIC disseminated intravascular coagulation                                    EDTA ethylenediaminotetraacetic acid                                          FN false negatives                                                            FP false positives                                                            Hyp hydroxyprolyl, Hyp represents 3Hyp (3-hydroxyprolyl)                       and/or 4Hyp (4-hydroxyprolyl)                                                INT iodonitrotetrazolium                                                      Leu leucyl                                                                    Lys lysyl                                                                     MA 4-methylcoumarinyl-7-amino                                                 MUG 4-methylumbelliferyl-β-D-glucuronide                                 MW molecular weight                                                           NA β-naphthylamino                                                       NADH hydrogenated nicotinamide adenine dinucleotide                           TNB tetrazolium nitroblue                                                     NPV negative predictive value                                                 NTC neotetrazolium chloride                                                   OM optical measurement                                                        Phe α-phenylalanyl                                                      Phg α-phenylglycyl                                                      pNA p-nitroanilino                                                            PPV positive predictive value                                                 Pro prolyl                                                                    RT room temperature (15-20° C.)                                        Sen sensitivity                                                               Spe specificity                                                               TN true negatives                                                             TP true positives                                                             TTC triphenyltetrazolium chloride                                             YNB nitrogen source marketed under the tradename "YEAST                        NITROGEN BASE"                                                             ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION

"Biological material" is understood here as meaning:

an aqueous human or animal body fluid, especially urine, blood, plasma,cerebrospinal fluid, pleural fluid, milk;

any type of water, especially running water (springs, rivers), stagnantwater (ponds, reservoirs, swimming pools), industrial water (tap water,sewage water, circulating water functioning in particular as heattransfer fluid or energy transfer fluid, waste water), drinking water;

a drink, especially originating from agri-foodstuffs and based on water,such as fruit juices, fizzy drinks, milk (mentioned above), wine, beer;

a food of animal and/or vegetable origin or a food preparation;

a plant extract, especially an aqueous, alcoholic or aqueous-alcoholicextract, particularly a pressed-out juice or an aqueous extract;

a solid or viscous product such as, in particular, pus, biopsies, feces,sputum, saliva, a plant, part of a plant, soil, sand; said product mayor may not contain water in its composition; and

man's environment in general (especially soil and water (mentionedabove) on the one hand and the ambient atmosphere on the other);examples are given below for evaluation of the bacteriological andmycological quality of the atmosphere in operating theater suites.

In brief, said biological material will be selected from the groupcomprising body fluids, foods, drinks and the products of theenvironment (including plants and their extracts).

In practice it is recommended, but not essential, that said biologicalmaterial be an aqueous product; for optimal implementation of thedetection method of the invention, it suffices for said biologicalmaterial to be converted (especially by grinding, dilaceration,extraction and/or addition of water) to a liquid sample, or already tobe in liquid form, in order to be tested. Said liquid sample willadvantageously be an aqueous liquid sample.

"Cell" is understood here as meaning any biological cell which isneither a bacterium nor a yeast. "Cellular material" is understood hereas meaning a material selected from (i) cells, (ii) fragments of saidcells (especially cell wall fragments), (iii) the content of said cellswhich belongs inside the wall, and (iv) mixtures thereof.

The biological material according to the invention in which it isdesired to determine the presence or absence of microorganisms, and itsliquid sample, can contain a cellular material.

By way of information, a patient's urine can contain on the one hand:

(A) in the case of a urinary infection, microorganisms such as:

Gram-negative bacteria, especially Eschetichia coli, Klebsiella (inparticular Klebsiella aerogenes, Klebsiella pneumoniae), Proteus (inparticular Proteus vulgaris, Proteus mirabilis), Pseudomonas (inparticular Pseudomonas aeruginosa), Serratia (in particular Serratiamarcescens) and/or Enterobacter (in particular Enterobacter aerogenes),

Gram-positive bacteria, especially Staphylococcus (in particularStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus), Streptococcus (in particular Streptococcus faecalis,Streptococcus faecium), Enterococcus and/or Corynebacterium, and/or

yeasts, especially Candida albicans, Candida glabrata (former name:Torulopsis glabrata), Candida tropicalis and/or Candida krusei, whichmay be pathogenic (these strains can induce septicemia and DIC); and onthe other hand:

(B) a cellular material comprising cells (in particular erythrocytes,leukocytes and/or epithelial cells), cellular enzymes (in particularurinary urokinase, leukocyte esterase and/or phosphatase), antibiotics(capable of interfering with or falsifying the detection of themicroorganisms present in the urine), peptides, proteins, pigmentsand/or cell wall fragments.

According to the invention, on the one hand the undissolved particles ofthe liquid sample of said biological material will be caused to migratethrough the gelled system so as to separate the microorganisms,according to their size, from the other undissolved solid componentsoriginating from said biological material, by centrifugation, and on theother hand said microorganisms will generally be grown in situ so thatthey can be detected.

"Reagent which induces a detectable variation in optical measurement"(alternative name: "colorimetric reagent") is understood here as meaninga product which, in the presence of a microorganism belonging to thegroup comprising bacteria and yeasts, causes a variation in OM which isassessed either with the naked eye or by means of a colorimeter. Inother words, the reagent which changes color in the presence ofmicroorganisms induces a color change in the visible spectrum or achange in fluorescence or luminescence, especially in the UV region.

Such a "colorimetric reagent" can be (i) a pH color indicator such asphenol red, (ii) a redox color indicator such as, in particular,dichloroindophenol, resazurin or a tetrazolium salt (for example TB,INT, TNB, NTC or TTC), (iii) a chromogenic substrate such asH-L-Ala-pNA, H-L-Leu-pNA, H-L-Phe-pNA, H-L-Pro-pNA, H-L-Lys-pNA orH-L-Hyp-pNA, or a fluorogenic substrate such as H-L-Ala-NA, H-L-Leu-NA,H-L-Phe-NA, H-L-Pro-NA, H-L-Lys-NA, H-L-Hyp-NA, H-L-Ala-MA, H-L-Leu-MA,H-L-Phe-MA, H-L-Pro-MA, H-L-Lys-MA, H-L-Hyp-MA or MUG phosphate, (iv) asubstrate cleavable by an osidase of the microorganisms, such asβ-D-glucosidase or, preferably, β-D-galactosidase, or (v) achemoluminescent substrate such as a luciferin compound.

The redox color indicators are mainly oxidizing compounds which revealthe presence of microorganisms by reaction with at least one reducingsubstance originating from said microorganisms, such as, in particular,NADH and dehydrogenases. The mechanism is analogous to that illustratedbelow for resazurin:

    resazurin+NADH→resorufin+NAD.sup.+

The chromogenic and fluorogenic substrates which are preferred accordingto the invention, namely H-L-Ala-pNA, H-L-Leu-pNA, H-L-Phe-pNA,H-L-Pro-pNA, H-L-Lys-pNA, H-L-Hyp-pNA, H-L-Ala-NA, H-L-Leu-NA,H-L-Phe-NA, H-L-Pro-NA, H-L-Lys-NA and H-L-Hyp-NA, are cleaved by theaminopeptidases produced by the microorganisms. Other peptide substratescleavable by said aminopeptidases, which are described in published PCTapplication WO-A-90/03726, can also be used in the present invention.

As indicated above, the gelled system according to the invention, whichis involved in step (1°) of the recommended method, comprises at leastone gelled layer, namely the "development phase", also designated hereby "first phase" or "first layer" for the sake of convenience. Thedevelopment phase consists of a polymeric substance (especially of thedextran or analogous type) swollen beforehand with water so as to form agel which retains in its bulk (or on its lower face in the case of thelargest particles), during centrifugation, the insoluble particlescontained in said liquid sample which have a size of between 0.5 and 8μm.

It is important that, in the swollen state, said polymeric substance ofthe development phase has a concentration by dry weight of 0.05 to 0.2g/ml (preferably a concentration of 0.1 g/ml) and is present (in thegel) in the form of swollen particles with a diameter of 90 to 320 μm,so that it retains in its bulk, or in the vicinity of its lower face,the bacteria (length: 0.5 to 5 μm; diameter: 0.1 to 2 μm) and themajority of the yeasts (diameter: 5 to 12 μm) but not the cells.

In practical terms, the polymeric substance of the development phasewill be swollen beforehand with the culture medium for growing themicroorganisms, said culture medium containing the above-mentionedcalorimetric reagent which induces a variation in OM.

For carrying out step (1°) of the recommended method, the gelled systemaccording to the invention can also comprise:

(b) a second, so-called absorption phase which is essentiallyimpermeable to the water which may be present in said liquid sample andto the substances dissolved therein, which retains in its bulk theinsoluble particles contained in said liquid sample which have a sizeless than 5 μm, but lets through the particles with a size greater thanor equal to 5 μm, during centrifugation, and which consists of (i) a gelor (ii) silica or silicate particles with a size less than 0.1 μm, saidsecond phase resting on said first phase, said intimate mixture beingessentially impermeable to the water which may be present in said liquidsample.

In one modified embodiment, the "absorption phase", "second phase" or"second layer" is a gel consisting of an intimate mixture of water andwater-absorbing polymer particles which have been swollen so that, insaid intimate mixture, said polymer particles have (α) a concentrationby dry weight of between 0.2 and 1 g/ml and (β) a diameter in theswollen state of between 160 and 530 μm.

In this case the absorption phase consists of a polymeric substance(especially of the dextran or analogous type) swollen beforehand withwater so as to form a gel, like said first phase, but so as to retain inits bulk (or on its lower face in the case of the largest particles),during centrifugation, on the one hand the insoluble particles containedin said liquid sample which have a smaller size of between 0.1 and 0.5μm, and on the other hand the insoluble particles which do not passthrough said first phase (i.e. with a size less than 5 μm).

In another modified embodiment, the absorption phase is a particulatelayer consisting of a mineral substance which on the one hand ismicronized so as to have a particle size less than 0.1 μm in the drystate, and on the other hand does not essentially swell in the presenceof water. This mineral substance will advantageously be selected fromthe group comprising silica (especially sand, quartz or glass) andsilicates such as aluminosilicates. Kaolin, a clay which does not swellin the presence of water, is more particularly recommended among thesilicates.

It has in fact been found that mineral substances such as silica andkaolin with a particle size less than 0.1 μm let the microorganismsthrough and retain the water and the dissolved substances which may bepresent in said liquid sample.

Thus the absorption phase will prevent the passage of the undissolvedsolid particles of said liquid sample such as the hemoglobin, thepigments (especially the urinary pigments when it is desired to detectthe presence or absence of microorganisms in the urine in the case of aurinary infection), the enzymes and the inhibitors, and will retain theundissolved particles such as the fragments of lyzed cells. By way ofexample, these fragments are retained after treatment with a lytic agentor in the presence of a lytic agent. This lytic agent can be adetergent, such as saponin, for lyzing the erythrocytes.

It is important that (1) in the swollen state, said polymeric substanceof the absorption phase has a concentration by dry weight of 0.2 to 1g/ml (preferably a concentration of 0.35 g/ml) and is present (in thegel) in the form of swollen particles with a diameter of 160 to 530 μm,and that (2) the mineral particles, such as micronized silica andkaolin, have a homogeneous diameter.

When it is present in said gelled system, said second phaseadvantageously contains one or more substances which inhibit anycontaminants or lyze the cells (especially the erythrocytes and/or theleukocytes) and/or eliminate the lipids (as in the clarification of milksamples to be tested) which are contained in said liquid sampleoriginating from said biological material and may interfere in step (4°)by reacting with said reagent which induces a detectable variation inoptical measurement in the presence of microorganisms.

When said second phase is not present in the gelled system of theinvention, it is recommended that said substance or substances (whichinhibit, lyze and/or eliminate) be included in said first phase.

For carrying out step (1°) of the recommended method, the gelled systemaccording to the invention can also comprise:

(c) a third, so-called barrier phase which cannot be crossed, during thecentrifuigation of step (3°), by any microorganisms present, said thirdphase being located underneath the first phase so that said first phaserests on said third phase.

Said third phase (or "third layer") is either a paraffin or a gel of apolymeric substance. When it is a paraffin, this is a solid paraffinwhich melts at a temperature above the multiplication temperature of themicroorganisms, for example:

a paraffin melting at a temperature above or equal to 30° C., when themicroorganism growth culture involved in step (4°) is carried out at RT,

a paraffin melting at a temperature above or equal to 45° C., when themicroorganism growth culture involved in step (4°) is carried out at 37°C., or

a paraffin melting at a temperature above or equal to 50-55° C., whenthe microorganism growth culture involved in step (4°) is carried out at45° C.

It will be preferable to use a paraffin melting at a temperature aboveor equal to 50° C., which will be introduced into the centrifuge tube inthe molten state.

When the barrier phase is a gel, said gel will consist of an intimatemixture of water and water-absorbing polymer particles which have beenswollen so that, in said intimate mixture, said polymer particles have(α) a concentration by dry weight of between 0.04 and 0.2 g/ml and (β) adiameter in the swollen state of between 30 and 130 μm.

Said gel consists of a polymeric substance (especially of the dextran oranalogous type) swollen beforehand with water so as to form a gel, likesaid first phase, but so as to retain in its bulk or on its upper face,during centrifugation, the insoluble particles contained in said liquidsample which have a larger size (i.e. a size greater than or equal to 5or 8 μm).

Thus the barrier phase will retain on its upper surface (in the case ofsolid paraffin) or in its bulk (in the case of gel) the cells,especially the erythrocytes with a mean size of 5-8 μm, the leukocyteswith a mean size of 10-12 μm, the epithelial cells (in particular in thecase of urine) with a mean size of 20-50 μm, the casts with a mean sizegreater than 40 μm, the crystals with a mean size of 20-200 μm, and thecell wall fragments (especially when the cells have been lyzed in theirpassage, during centrifugation, through the first phase or the combinedsecond phase/first phase).

In practical terms, whatever the nature of the barrier phase, the solidparticles contained in said liquid sample will spread out according to agradient, with the smallest (i.e. the lightest) on top of the largest(i.e. the heaviest).

When the gelled system does not comprise the barrier phase, it isimportant that said gelled system be placed in a centrifuge tube with aconical bottom (as envisaged in the documents FR-A-2 577 321 and EP-A-0454 509 cited above) or a bottom provided with a capillary. In thiscase, the heaviest solid particles spread out at the bottom of thecentrifuge tube, as indicated above, with the microorganisms just abovethe cells.

It is important that, in the swollen state, said polymeric substance ofthe gel of the barrier phase has a concentration by dry weight of 0.04to 0.2 g/ml (preferably a concentration of 0.14 g/ml) and is present inthe form of swollen particles with a diameter of 30 to 130 μm.

When the barrier phase is present in the gelled system according to theinvention, said gelled system can be accommodated in any type ofcentrifuge tube, especially a tube with a round or spherical bottom.

The presence of the absorption phase is advantageous in that it makes itpossible to prevent the liquid (mainly the water) of said liquid samplefrom coming into contact with the development phase before and aftercentrifugation.

The simultaneous presence of the absorption phase and the barrier phaseis advantageous in that it enables a development phase of smallthickness to be used. Consequently, the microorganisms present in thebulk or on the lower face of said development phase will be moreconcentrated in a given volume and hence more readily detectable duringthe calorimetric development (either with the naked eye or by means of acolorimeter).

The gelled system according to the invention which comprises said firstphase, either on its own or associated with at least one of said secondand third phases, is impermeable to the liquid of said liquid sample.During centrifugation, said liquid and the ingredients dissolved thereindo not pass through said gelled system; on the other hand, theundissolved ingredients of said liquid sample migrate into the thicknessof said gelled system and are separated according to their size, asexplained above.

For carrying out step (1°) of the recommended method, the gelled systemaccording to the invention can also comprise:

(d) a fourth, so-called protection phase which (i) is an inert oil witha density less than that of water, preferably a paraffin oil, (ii)before centrifugation, rests on said first phase in the absence of thesecond phase, or rests on said second phase when the latter is presentin said gelled system, and (iii) after centrifugation according to step(3°), rests on the water of said liquid sample.

The oil of the protection phase has to be inert towards the constituentsof the gelled system and the components of the liquid sample. An oilwhich melts at a temperature below RT and has a boiling point above 50°C., or even above 55° C., is more particularly recommended. A paraffinoil is particularly suitable. In practical terms, a volume of 0.25 to 1ml of the protection phase will be introduced into a 5 ml centrifugetube.

According to the invention, the protection phase advantageously servestwo purposes: on the one hand it prevents dehydration and contaminationof the gelled system during storage, and on the other hand it means thatthe layer of said gelled system on which it rests is not degraded whensaid liquid sample is poured into the centrifuge tube.

It is preferentially recommended to use in step (1°) either a gelledsystem comprising, from top to bottom, in a conical-bottomed centrifugetube:

said fourth phase,

said second phase and

said first phase,

or a gelled system comprising, from top to bottom, in a round-bottomedor spherical-bottomed centrifuge tube:

said fourth phase,

said second phase,

said first phase and

said third phase,

or a gelled system comprising, from top to bottom, in acapillary-bottomed centrifuge tube:

if appropriate, said fourth phase and/or said second phase, and

in the capillary, said first phase.

The centrifugation of step (3°) is carried out above 500 g and in therange 1000 g to 5000 g (i.e. approximately at an angular velocity of2200 to 4500 rpm when using the customary centrifugation devices). Theduration of this centrifugation is at least 5 minutes and preferably 10to 30 minutes. The centrifugation according to the invention willadvantageously be carried out at 2000-4000 g for 15 minutes.

Said centrifugation according to the invention is generally carried outat RT. If required, said centrifugation can be carried out at atemperature between RT and 45° C. (preferably between RT and 37° C.),i.e. at a temperature analogous to that of the culture envisaged in step(4°) for growing the microorganisms which may be present in said liquidsample, but it is not essential to use a temperature above RT during thecentrifugation.

When the protection phase is present in the gelled system of theinvention, the liquid of said liquid sample migrates through saidprotection phase during the centrifuigation; after said centrifugation,said liquid, devoid of the undissolved solid constituents which havealso migrated, is situated between said protection phase and theabsorption phase (if the latter is present) or the development phase (inthe absence of the absorption phase in said gelled system).

The development envisaged in step (4°) takes place on the one handduring centrifugation and on the other hand, if appropriate, afterculture of the microorganisms at a temperature between RT and 45° C. Theduration of the culture required for growing the microorganismsgenerally depends on the initial concentration of said microorganisms(bacteria and/or yeasts) in said liquid sample.

In practice, for a liquid sample with an initial bacteria concentrationbelow the detection threshold (i.e. 10³ germs/ml), it is necessary toanticipate a culture time of at most 24 h (preferably 1-2 h), except inthe case of Staphylococcus aureus strains, which multiply relativelyslowly (for said Staphylococcus strains, a culture time of about 24 hmust be expected).

For a liquid sample with an initial yeast concentration below thedetection threshold (i.e. 10³ germs/ml), it is necessary to anticipate aculture time of at most 36 h (preferably 24-30 h). To favor thedetection of said yeasts, it is recommended to incorporate anantibacterial agent, such as that described in the documentWO-A-90/03726 cited above, especially gentamycin, into the absorptionphase and/or the development phase in order to inhibit bacterial growthor destroy said bacteria.

The culture medium used to swell the development phase comprises acarbon source, a nitrogen source and trace elements, for example apeptone (5 to 20 g/l), a sugar (1 to 20 g/l, preferably 5 g/l), a yeastextract (1 to 5 g/l) and vitamins (10 to 20 ml), with a buffer forstabilizing the pH at a value of between 6.0 and 8.0. For bacteria, thismedium consists for example of a heart-brain broth with a sugar added.For yeasts, this medium can be either identical to that for bacteria, ordifferent, for example a YNB+glucose medium, a Sabouraud medium or aYNB+maltose medium (which are described in WO-A-90/03726 cited above).

For Gram-negative bacteria, the culture medium used will advantageouslybe heart-brain broth+sugar containing the following as the preferredreagent which induces a detectable variation in optical measurement:

resazurin at a final concentration of 0.05 g/ml (0.2 mM),

phenol red at a final concentration of about 0.065 g/ml (0.17 mM),

the chromogenic substrate H-L-Ala-pNA at a final concentration of about0.50 g/l (2 mM), or

the fluorogenic substrate H-L-Ala-NA (about 2 mM), this medium beingbuffered at pH 6.5-8.0 by means of a phosphate or Tris buffer (0.1 M)and it being possible for this medium to contain the following ifappropriate:

an activator belonging to the group comprising divalent cations,especially Mg²⁺, Ca²⁺ or Mn²⁺ (for example a solution of MnCl₂ or MnSO₄at about 30 mg/l),

a diazotization reagent such as ρ-dimethylaminocinnamaldehyde or FastBlue BB, and/or

one or more inhibitors of the secondary reactions associated especiallywith the presence of enzymes in said liquid sample to be tested (forexample, in the case of urine screening, aprotinin to inhibit theurinary urokinase and EDTA to inhibit the phosphatases).

In practice, the inhibitor or inhibitors can be placed in thedevelopment phase and/or the absorption phase.

For Gram-positive bacteria, the culture medium used will advantageouslybe the medium described above for Gram-negative bacteria, with thefollowing as the preferred reagent which induces a detectable variationin optical measurement:

resazurin at a final concentration of about 0.05 g/ml,

the chromogenic substrate H-L-Leu-pNA at a final concentration of about2 mM, or

the fluorogenic substrate H-L-Leu-NA at a final concentration of 2 mM,

the pH being buffered at 6.0-8.0 and it being possible for said mediumto contain, if appropriate, an activator, a diazotization reagent and/orone or more inhibitors, as indicated above.

For yeasts, the culture medium used will advantageously be a YNB+sugarmedium buffered at pH 6.5-8.0, with the following as the preferredreagent which induces a detectable variation in optical measurement: thechromogenic substrate H-L-Pro-pNA or an equivalent substrate, anessential inhibitor being an antibiotic, such as gentamycin,streptomycin or chloramphenicol, for inhibiting bacterial growth. Saidessential inhibitor can also be present in the absorption phase.

Other advantages and characteristics of the invention will be understoodmore clearly from the following description of the drawings andExamples. Of course, these data as a whole in no way imply a limitation,but are given by way of illustration.

The gelled system 10 according to the invention is accommodated in acentrifuge tube with a conical bottom, 6a, a spherical bottom, 6b, or abottom provided with a capillary, 6c. Said gelled system 10 comprises atleast one layer, namely the development phase 1, which is a gel swollenbeforehand by means of an aqueous microorganism culture medium, and, ifappropriate, one or more other layers, namely the absorption phase 2,which is either a gel swollen beforehand with water and capable ofcontaining one or more inhibitors (such as aprotinin and EDTA), or amicronized particulate layer (silica or kaolin) with a particle sizeless than 0.1 μm, the barrier phase 3, which is a gel swollen beforehandwith water or a layer of paraffin that is solid under thecentrifuigation and culture conditions, and/or the protection phase 4,which is a layer of paraffin that is liquid under said conditions.

FIG. 1 relates to a gelled system 10 according to the inventioncomprising a single layer, the development phase 1, accommodated in aconical-bottomed centrifuge tube 6a. The liquid sample 5 is poured ontop of said development phase 1. On centrifuigation, the undissolvedparticles of said liquid sample migrate into the thickness of thedevelopment phase; on the other hand, the liquid of said liquid sample 5and the dissolved ingredients do not pass through the development phase.After said centrifugation, the largest particles, which are theheaviest, are spread out at the bottom of the tube 6a according to theirsize, with the cells at the bottom and, above said cells, themicroorganisms, which are located in the vicinity of the lower part ofthe development phase 1.

The gelled system according to FIG. 1 gives good results, but it is nottotally satisfactory in that the gel of the development phase 1 (i) isin contact with said liquid sample 5 during centrifugation and culture,and (ii) can consequently be diluted by said liquid sample 5.

FIGS. 2a and 2b relate to the same gelled system 10 beforecentrifugation (FIG. 2a) and after centrifugation (FIG. 2b). Comparedwith the gelled system of FIG. 1, the gelled system of FIGS. 2a and 2balso comprises a layer of oil (liquid paraffin) constituting theprotection phase. The development phase 1 is poured into the tube 6a,followed by the protection phase 4, and said gelled system 10 iscompressed by centrifugation (2000-3000 g for 10-20 minutes). The liquidsample to be tested is then poured into the tube 6a. Said liquid sample5 spreads out on top of the protection phase 4 (cf FIG. 2a) beforecentrifugation.

During centrifugation, said liquid sample 5 migrates through theprotection phase 4 together with the soluble ingredients it contains.Its solid particles migrate through the development phase 1, theheaviest of said particles spreading out at the bottom of the tube asindicated above for FIG. 1. After centrifugation, the liquid of theliquid sample 5 is located between the protection phase 4 and thedevelopment phase 1 (cf. FIG. 2b).

Although the gelled system of FIGS. 2a and 2b is admittedly better thanthat of FIG. 1, it nevertheless has the disadvantage that the liquidsample 5 is in contact with the development phase 1 after centrifugationduring the so-called culture step.

FIG. 3 relates to a gelled system 10 according to the inventioncomprising two layers: the development phase 1 and the absorption phase2. The development phase 1 is poured into the conical-bottomedcentrifuge tube 6a, followed by the absorption phase 2, and said gelledsystem 10 is compressed by centrifugation (1000-3000 g for 10-20minutes). The liquid sample 5 to be tested is then poured into the tube6a. Said liquid sample 5 spreads out on top of the absorption phase.

During the centrifugation of step (3°), the solid particles contained inthe liquid sample 5 migrate through the absorption phase 2 and/or thedevelopment phase 1 according to their size; on the other hand, theliquid of said liquid sample 5 and the ingredients dissolved therein donot pass through the absorption phase 2.

After the centrifuigation of step (3°), the largest and heaviestparticles of said liquid sample 5 are found at the bottom of the tube6a, spread out as indicated above for FIGS. 1 and 2a-2b.

It is advantageous on the one hand that the development phase 1 isisolated from the liquid of said liquid sample 5 by the absorption phase2 after the centrifugation of step (3°), and on the other hand that theabsorption phase 2 absorbs the small particles.

FIG. 4a [before centrifugation] and FIG. 4b [after centrifugationaccording to step (3°)] relate to a gelled system 10 according to theinvention which is analogous to that of FIG. 3 but also comprises alayer of oil (paraffin oil), namely the protection phase 4, in aconical-bottomed centrifuge tube 6a. The development phase 1 is pouredinto the tube 6a, followed by the absorption phase 2 and then theprotection phase 4, and said gelled system 10 is compressed bycentrifugation (1000-3000 g for 10-20 minutes). The liquid sample 5 tobe tested is then poured into the tube 6a. Said liquid sample 5 spreadsout on top of the protection phase 4 (cf FIG. 4a).

During the centrifugation of step (3°), the liquid sample 5 migratesthrough the protection phase 4 and the solid particles it contains alsomigrate through the absorption phase 2 and/or the development phase 1according to their size; on the other hand, the liquid of said liquidsample 5 and the ingredients dissolved therein do not pass through theabsorption phase 2. After centrifugation, the largest and heaviestparticles are spread out at the bottom of the tube 6a as indicatedabove.

FIG. 5 relates to a gelled system 10 according to the inventioncomprising three layers, namely, from bottom to top: the barrier phase3, the development phase 1 and the absorption phase 2, the layer 1 beinga gel, the layer 2 being either a gel or a particulate phase and thelayer 3 being either a gel or a solid paraffin.

The barrier phase 3 (melted beforehand if it consists of a paraffinwhich is solid at a temperature between RT and 50-55° C., and thencooled), the development phase 1 and then the absorption phase 2 arepoured successively into a spherical-bottomed tube 6b. Said gelledsystem 10 is compressed by centrifugation (1000-3000 g for 10-20minutes). The liquid sample 5 to be tested is then poured into the tube6b. Said liquid sample 5 spreads out on top of the absorption phase 2before the centrifugation of step (3°).

During the centrifugation of step (3°), the liquid of said liquid sample5 and the dissolved constituents it contains do not migrate through theabsorption phase 2; only the solid particles of said liquid sample 5migrate, according to their size, through the gelled system 10consisting of the combined absorption phase/development phase/barrierphase. When the barrier phase 3 is a solid paraffin, all the large andheavy particles do not pass through its thickness but spread out at thedevelopment phase/barrier phase interface according to their size.

The configuration of the layers 2, 1 and 3 according to FIG. 5 (the sameapplies to the gelled system of FIGS. 6a and 6b) offers the advantage ofbeing able to reduce the thickness of the development phase 1 sandwichedbetween the absorption phase 2 and the barrier phase 3, and thus ofimproving the reading of the variation in OM, either with the naked eye(color change) or by means of a calorimeter.

FIG. 6a [before the centrifugation of step (3°)] and FIG. 6b [after thecentrifugation of step (3°)] relate to a gelled system 10 according tothe invention which is analogous to that of FIG. 5 but also comprises alayer of oil 4 (paraffin oil) constituting the protection phase. Thesolid particles of the liquid sample 5 spread out as indicated for FIG.5, the protection phase 4 enabling said liquid sample 5 to migrateduring the centrifugation of step (3°).

The polymeric substances used in the swollen state in the gels (from topto bottom) of the absorption phase 2, the development phase 1 and thebarrier phase 3 are advantageously dextran particles. Particularlysuitable products are those of the Sephadex® G range marketed byPHARMACIA FINE CHEMICALS (Uppsala, Sweden), namely:

for the absorption phase 2, the product G 25C,

for the development phase 1, the products G25 to G 100 of so-calledcoarse to medium particle size, such as G 25M, G 50C, G 50M, G 75 and G100 (the preferred product being G 100),

for the barrier phase 3, the products G 150 to G 200 of so-called mediumto superfine particle size, such as G 150, G 150S, G 200 and G 200S (thepreferred products being G 200 and especially G 200S).

The characteristics of these products of the dextran type are collatedin Table I below.

                  TABLE I                                                         ______________________________________                                                                   Fractionation range (MW)                           Phase    Gel     (A)       (B)      (C)                                       ______________________________________                                        Absorption                                                                             G 25C   100-300   1000-50000                                                                             100-5000                                     G 25M 50-150 1500-30000 500-10000                                             G 50C 100-300 1500-30000 500-1000                                            Development G 50M 50-150 1500-30000 500-10000                                  G 75 40-120 3000-80000 1000-50000                                             G 100 40-120 4000-150000 1000-100000                                          G 150 40-120 5000-300000 1000-150000                                         Barrier G 150S 10-40 5000-150000 1000-150000                                   G 200 40-120 5000-600000 10000-200000                                         G 200S 10-40 5000-250000 10000-200000                                      ______________________________________                                         Notes:                                                                        (A): diameter in μm of the particles in the dry state                      (B): globular peptides and proteins                                           (C): dextrans                                                                 G 25C: "Sephadex ® G 25 Coarse                                            G 25M: "Sephadex ® G 25 Medium                                            G 50C: "Sephadex ® G 50 Coarse                                            G 50M: "Sephadex ® G 50 Medium                                            G 75: "Sephadex ® G 75                                                    G 100: "Sephadex ® G 100                                                  G 150: "Sephadex ® G 150                                                  G 150S: "Sephadex ® G 150 Superfine                                       G 200: "Sephadex ® G 200                                                  G 200S: "Sephadex ® G 200 Superfine                                  

The characteristics relating to the particle size in the swollen stateand to the concentration, which are important according to theinvention, have been given for the preferred gels in Tables II and IIIbelow.

                  TABLE II                                                        ______________________________________                                                        Diameter of the particles (μm) in the swollen state        Phase   Gel     (A)            (B)                                            ______________________________________                                        Absorption                                                                            G 25C   160-530        172-516                                          Development G 100 90-320 103-311                                              Barrier G 200S 30-130 32-129                                                ______________________________________                                         Notes:                                                                        (A): range of the invention                                                   (B): preferred range                                                     

                  TABLE III                                                       ______________________________________                                                            Concentration (g/ml)                                      Phase       Gel     (A)         (B)  (C)                                      ______________________________________                                        Absorption  G 25C   0.2         0.35 1                                          Development G 100 0.05 0.1 0.2                                                Barrier G 200S 0.04 0.14 0.2                                                ______________________________________                                         Notes:                                                                        (A): minimum concentration according to the invention                         (B): preferred concentration according to the invention                       (C): maximum concentration according to the invention                    

The following comments should be made with reference to the minimum andmaximum concentrations of the gels shown in Table III:

(1) if the gel in contact with the liquid of the liquid sample 5 (i.e.layer 1 of FIG. 1 or layer 2 of FIGS. 3 and 5) is more concentrated,there is a risk that said liquid sample 5 will pass through said gelduring centrifugation;

(2) if all the gels are more concentrated, this seriously interfereswith the detection of the presence or absence of microorganisms;

(3) if at least one of the gels is more dilute, exchanges of liquid cantake place on the one hand between the liquid sample and the underlyinggel, or on the other hand between two adjacent gels, thereby falsifyingsaid detection.

It is possible to omit the barrier phase 3, as illustrated in FIGS. 1,2a-2b, 3 and 4a-4b, when the large particles (erythrocytes, leukocytes,other cells) have no activity on the reagent which induces a detectablevariation in optical measurement. This is particularly the case whensaid reagent is resazurin.

It is possible to omit the absorption phase 2 and the barrier phase 3,as illustrated in FIGS. 1 and 2a-2b, when no substance (dissolved orparticulate) present in the liquid sample 5 has any activity on saidreagent which induces a detectable variation in optical measurement, orwhen any possible interference can be inhibited during the developmentof step (4°).

If the concentration of the microorganisms which may be present in theliquid sample 5 to be tested, and which have migrated during thecentrifugation of step (3°) to end up in the development phase 1 (eitherin the thickness of said phase 1 or on top of the interface betweenphase 1 and phase 3 or between phase 1 and the lower wall of the tube6a), is above the detection threshold of the reagent of the developmentphase 1 which induces a detectable variation in optical measurement, themultiplication of the microorganisms by culture in situ in step (4°) isnot necessary.

On the other hand, if after said centrifugation the concentration of themicroorganisms is below the detection threshold, it is necessary tocarry out said multiplication by culture in situ in said developmentphase 1 in the centrifuge tube, preferably at a temperature of 37° C.

The centrifuge tube 6a, 6b or 6c is made of a transparent material(plastic or glass). The centrifuge tube which can be used according tothe invention will preferably be made of plastic and produced bymolding. In a modified embodiment, said tube can be transparent over atleast part of its peripheral surface, at the level of the zone packedwith the development phase 1.

According to the invention, the detection threshold of themicroorganisms, based on the initial concentration of saidmicroorganisms in said liquid sample 5, is 10³ germs/ml, i.e. markedlylower than the threshold of 10⁵ germs/ml of the techniques of the priorart, illustrated especially by the above-mentioned Bac-T-Screen® andFiltraCheck®-UTI tests.

This improvement is due to the fact that, according to the invention,(α) the microorganisms of the liquid sample 5 which have migrated duringcentrifugation are concentrated in the development phase 1, and then (β)if appropriate, they are multiplied in the nutrient medium.

To concentrate the microorganisms in the development phase, it isrecommended according to the invention to use a development phase 1 withthe smallest possible volume and thickness. The shape of the centrifugetube will consequently be chosen so as to maximize the concentrationfactor of the microorganisms, which is directly proportional to theratio of the volume of the test sample to the volume of the developmentphase. The volume of the development phase is determined by the formulaV=πR² h (where V is expressed in ml and the radius R and height h areexpressed in cm). The smaller the diameter of the tube, the more theconcentration factor increases. By way of example, the concentrationfactor can be a multiple of 10³ when the radius of the tube is dividedby 10.

The capillary of the tube 6c allows a good concentration of themicroorganisms in the development phase.

FIGS. 7 and 8 show preferred gelled systems according to the invention,as they appear after centrifugation. The tube of FIG. 7, which hassimilar contents to the tube of FIG. 4b, comprises a development phase 1whose volume is smaller than those of the phase 2 and the sample 5 andwhich is accommodated at the bottom of said centrifuge tube 6a in thelower portion of the conical part.

The tube 6b of FIG. 8, which has similar contents to the tube of FIG.6b, has a spherical bottom (whose diameter can be smaller than that ofthe rest of the tube) accommodating the barrier phase 3 and thedevelopment phase 1. This bottom is such that the development phase 1sandwiched between the lower barrier phase 3 and the upper absorptionphase 2 has the smallest possible volume and thickness.

By way of example, if a transparent plastic tube according to FIG. 8 isused which has a volume of 12 ml and an internal diameter of 1.35 cm andinto which a volume of 6 ml of urine sample to be tested is introduced,to obtain the development phase 1 in the form of a 0.2 cm high ring, thevolume of said phase 1 to be used must have a value V ofπ.(1.35/2)².0.2, i.e. about 0.3 ml. The concentration factor (F) of themicroorganisms in the urine is then 6/0.3=20 for a colored ring to bevisible. As the positive reaction starts with a spot representing lessthan a tenth of the diameter of the tube, the useful reaction volume ofthe development phase 1 then becomes 0.003 ml, which leads to aconcentration factor of 6/0.003=2000, i.e. the sensitivity threshold ofthe concentration factor increases by a multiple of 10³, as mentionedabove. In a modified embodiment, when the cross-section of the lowerpart of the tube is reduced by a factor of 10 (i.e. divided by 10 togive a diameter of 0.135 cm), V becomes 0.3×10⁻² ml, i.e. the factor Fincreases by 10² for a ring and by 10³ for a spot.

FIGS. 9 and 10 each show a centrifuge tube 6c made of a transparentplastic (for example polystyrene or polycarbonate), which can be usedaccording to the invention. The tube 6c offers the advantage that themicroorganisms present in the development phase accommodated in thecapillary can be counted.

According to FIG. 8, the tube 6c is provided at its lower end with acapillary 7 located along the axis of said tube, said capillary beingintended to receive the development phase (reference 1 above). Thecylindrical part of the tube 6c is joined to the mouth of the capillary7 via a frustoconical portion 20. The development phase is accommodatedin the capillary tube up to the line 22, which corresponds to theintersection of the frustoconical portion 20 with the mouth of thecapillary 7. If appropriate, the protection phase (reference 4 above)and/or the absorption phase (reference 2 above) are accommodated abovethe line 22. The outer cylindrical wall of the tube 6c extendsdownwards, facing the capillary, in the form of two legs (or extensions)8 and 9, which are approximately diametrically opposite one another andprovide the tube with stability when it is upright. These legs 8 and 9form a base; two windows are created between them and enable thecontents of the capillary 7 to be observed with the naked eye or bymeans of an optical device (calorimeter, spectrometer, etc.), beforecentrifugation and then after centrifugation or culture, so that thebacteria or yeasts present can be counted.

The tube 6c of FIG. 10 is made on the one hand so that the bacteria oryeasts present can be counted, as indicated above for the tube of FIG.9, and on the other hand so that said bacteria or yeasts present can becollected for identification. This tube comprises a capillary 7, afrustoconical wall 20 and an inner cylindrical wall 17, the line 22corresponding to the intersection of the surface 20 with the mouth ofthe capillary 7. The mouth of the cylindrical portion 17 is eitherparallel to the axis of the tube 6c or, as shown by 11, funnel-shaped.

In the vicinity of the upper end, the outer surface of the tube 6c ofFIG. 10 has one or more grooves 14 and/or 15 capable of accommodating anO-ring seal (especially made of rubber) not shown here, one of thesegrooves, 14, being located between two flanges or lips 12 and 13 and theother groove, 15, being located between the flange 13 and the outersurface 16 of the tube. An annular abutment 18 is provided on said outersurface 16. The assembly comprising the flange 12 and/or 13 and theouter surface 16 is intended to be sealed with a cylindrical cap,especially a plastic cap, covering the outer upper part of the tube 6cas far as the abutment 18. The legs or extensions 8 and 9 act as a basefor keeping the centrifuge tube 6c upright, as indicated above for thetube of FIG. 9. A portion of the outward facing surface of one of theselegs, 8, can be flat so that information can be written or stuck on saidleg if appropriate. The legs 8 and 9 can also have a set of severalalternating grooves 19 and lips 21 on their outward facing surface sothat, if necessary, the tube 6c of FIG. 10 (which can have a very smallheight, in particular 30 mm) can be housed in the mouth of another(larger) centrifuge tube suitable for the centrifugation device used.

The bacteria or yeast present in the development phase accommodated inthe capillary 7 up to the height of the line 22 are observed and countedby means of the windows created between the legs 8 and 9, as indicatedabove for the tube of FIG. 9.

To collect the microorganisms present in the development phaseaccommodated in the capillary 7 of the tube of FIG. 10, (1°) said tubeis inverted to discard the supernatant, said development phase remainingin the capillary, (2°) an O-ring seal is placed in the groove 14, (3°)the assembly comprising the flange 12, the seal housed in the groove 14,the flange 13 and the surface 16 is inserted inside the mouth of asecond centrifuge tube, containing the liquid recovery medium, until thesecond centrifuge tube comes into contact with the abutment 18, the tube6c being kept upside down (i.e. with the capillary 7 pointing upwards),(4°) the two centrifuge tubes joined together in this way by theirmouths are shaken, and then (5°) they are centrifuged at 1000-2000 g for2-5 minutes to transfer the development phase, initially contained inthe capillary 7, into the recovery liquid, which is at the bottom of thesecond tube.

This procedure enables the microorganisms present to be recoveredeasily, without contamination and substantially without loss. Thistechnique is much more efficient than that which consists in removingthe development phase accommodated in the capillary 7 by means ofsuction with a syringe; in fact, the swollen polymer particles of thegel constituting the development phase do not easily pass through thesyringe needle because of their dimensions.

In a modified embodiment, the bulk of the development phase containingmicroorganisms can be collected by using the tube of FIG. 9. To do this,(1a) the supernatant situated above the line 22 is discarded, (2a) anaqueous washing solution is added and the tube 6c is sealed and shaken,(3a) the tube is centrifuged at 1000-2000 g for 2-5 minutes, (4a)operations (2a) and (3a) are repeated until the supernatant iscolorless, and (5a) the resulting supernatant is discarded, an aqueousrecovery solution is added and the tube is shaken to collect theresulting liquid medium containing the bulk of the development phase andhence most of the microorganisms present.

From a practical point of view, so that the development phase remains inthe capillary when the tube 6c is inverted before shaking and thencentrifugation, it is important for the internal diameter of thecapillary to be less than or equal to 2.5 mm. An appropriate internaldiameter is between 1.2 and 2.3 mm. Very good results have been obtainedwith internal diameters of 2.0 and 2.2 mm.

To improve the sensitivity of the detection of the microorganisms andachieve a good recovery of the development phase, it is recommended tosatisfy at least one, and preferably all, of the following conditions:

A- the internal diameter of the capillary is less than 2.5 mm,

B- the angle α of the frustoconical portion 20 is less than or equal to30° (i.e. ≦π/6), that is to say that the generatrix of thisfrustoconical portion is slightly inclined relative to the vertical axisof the tube (angle of inclination less than or equal to α/2),

C- the volume of the capillary is less than or equal to 0.6 ml andpreferably less than or equal to 0.1 ml.

If condition B or condition C is not satisfied, there is a risk thattraces of development phase, and hence microorganisms, may be depositedon the inner wall of the tube, especially above the region of theintersection 22.

EXAMPLE 1

The following are introduced successively into a transparent plasticcentrifuge tube with a volume of 5 ml and an internal diameter of 1.10cm:

0.5 ml of a gel of G 200S/distilled water, in which the G 200S has aconcentration by dry weight of 0.2 g/ml and a particle size in theswollen state of 40-100 μm, to form the barrier phase;

0.25 ml of a gel of G 100/aqueous culture medium, in which the G 100 hasa concentration by dry weight of 0.2 g/ml and a particle size in theswollen state of 120-300 μm, said aqueous culture medium having thefollowing composition:

    ______________________________________                                                 heart-brain broth                                                                             37 g/l                                                 glucose 5 g/l                                                                 agar (type IX) 0.33 g/l                                                       pH 7.4 ± 0.1                                                             ______________________________________                                    

and containing 0.05 g/ml of resazurin, to form the development phase;and

0.5 ml of a gel of G 25C/distilled water, in which the G 25C has aconcentration by dry weight of 1 g/ml and a particle size in the swollenstate of 40-110 μm, to form the absorption phase.

The resulting gelled system is compressed by centrifugation (1000-3000 gfor 10-20 minutes at RT). In a modified embodiment, said compressingcentrifugation can be carried out after the introduction of each of saidphases and before the introduction of the next phase.

The gelled system obtained by this process is ready to use.

EXAMPLE 2

1 ml of paraffin oil is introduced as a protection phase on top of thegelled system prepared according to Example 1 above.

This gives a device for detecting the presence or absence ofmicroorganisms in a sample of a biological material, said device beingstable on storage.

EXAMPLE 3

The following are introduced successively into a transparent plasticcentrifuge tube with a volume of 5 ml and an internal diameter of 1.10cm:

0.5 ml of a paraffin melting at a temperature above or equal to 50° C.(this paraffin being introduced in the molten state), to form thebarrier phase (after cooling to RT);

0.25 ml of a gel of G 100/aqueous culture medium, in which the G 100 hasa concentration by dry weight of 0.2 g/ml and a particle size in theswollen state of 103-311 μm, said aqueous culture medium having thefollowing composition:

    ______________________________________                                                 heart-brain broth                                                                             37 g/l                                                 glucose 5 g/l                                                                 agar (type IX) 0.33 g/l                                                       pH 7.4 ± 0.1                                                             ______________________________________                                    

and containing 2 mmol/l of H-L-Ala-NA in phosphate buffer (0.1 M) at pH8.0, to form the development phase; and

0.5 ml of a gel of G 25C/distilled water, in which the G 25C has aconcentration by dry weight of 1 g/ml and a particle size in the swollenstate of 32-129 μm, the distilled water used to produce this gelcontaining 0.06 g/ml of EDTA, to form the absorption phase.

The resulting gelled system is compressed by centrifugation (1000-3000 gfor 10-20 minutes at RT). In a modified embodiment, said compressingcentrifugation can be carried out after the introduction of each of saidphases and before the introduction of the next phase.

The gelled system obtained by this process is ready to use.

EXAMPLE 4

0.25 ml of paraffin oil is introduced as a protection phase on top ofthe gelled system prepared according to Example 3 above.

This gives a device for detecting the presence or absence ofmicroorganisms in a sample of a biological material, said device beingstable on storage.

EXAMPLE 5

The procedure is as indicated in Example 3 except that the aqueousculture medium used for swelling the G 100 to obtain the developmentphase also contains the diazotization reagentρ-dimethylaminocinnamaldehyde.

EXAMPLE 6

The following are introduced successively into a conical-bottomedtransparent plastic centrifuge tube with an internal diameter (in itscylindrical portion) of 1 cm and a volume of 5 ml:

0.20 ml of a gel of G 100/aqueous culture medium, in which the G 100 hasa concentration by dry weight of 0.2 g/ml and a particle size in theswollen state of 103-311 μm, said aqueous culture medium having thefollowing composition:

    ______________________________________                                               YNB            6.7    g/l                                                maltose 20.0 g/l                                                              gentamycin 0.05 g/l                                                           cycloheximide 0.5 g/l                                                         pH 6.0                                                                      ______________________________________                                    

and containing 1 mmol/l of H-L-Pro-pNA (this culture medium containing asubstrate specific for strains of Candida albicans so that said Candidaalbicans can be distinguished from the other Candida), to form thedevelopment phase; and

0.5 ml of micronized kaolin with a homogeneous particle size of0.055-0.060 μm, to form the absorption phase.

The resulting gelled system is compressed by centrifugation at 2000 gfor 15 minutes at RT.

The gelled system obtained by this process is ready to use and isintended specifically for the identification of Candida albicans among agroup of non-isolated strains of Candida.

EXAMPLE 7

1 ml of paraffin oil is introduced on top of the gelled system preparedaccording to Example 6 in order to form the protection phase, and theresulting gelled system is compressed by centrifugation at 2000 g for 15minutes at RT.

EXAMPLES 8 AND 9

The procedure is as indicated in Example 6 above except that 0.20 ml ofthe nutrient medium of Example 1, containing resazurin, is used as thenutrient medium to obtain the development phase for the preparation of agelled system specific for the detection of bacteria (Example 8). Theresulting gelled system is used to prepare, according to Example 7, aready-to-use gelled system comprising the protection phase (Example 9).

EXAMPLES 10 AND 11

The procedure is as indicated in Example 6 above except that 0.20 ml ofa nutrient medium specific for strains of Aspergillus is used as thenutrient medium to obtain the development phase for the preparation of agelled system specific for the detection of these yeasts (Example 10).The resulting gelled system is used to prepare, according to Example 7,a ready-to-use gelled system comprising the protection phase (Example11).

EXAMPLE 12

A volume of 1 to 4 ml of sterilized urine (into which differentconcentrations of strains of Escherichia coli had been introduced), orof urine from a patient suffering from a urinary infection caused bystrains of Escheridia coli, was introduced into the tubes containing thegelled systems prepared according to Examples 1 and 5. Aftercentrifugation at 3000-5000 g for 20 minutes at RT in the case of aninfected urine sample containing strains of Escheridia coli at aconcentration of 10⁵ CFU/ml, a pink spot is observed with resazurin(i.e. the gelled system prepared according to Example 1) and a red spotis observed with the mixture H-L-Ala-NA/diazotization reagent (i.e. thegelled system prepared according to Example 5). These colored spotslocated in the development phase spread and each form a colored ringafter a few hours at RT or 37° C.

The results obtained with resazurin have been collated in Table IVbelow.

                  TABLE IV                                                        ______________________________________                                        Detection of strains of Escherichia coli with resazurin                                    Bacteria/ml in infected urine                                    Reading  10.sup.8                                                                              10.sup.7                                                                              10.sup.6                                                                            10.sup.5                                                                            ≦10.sup.4                                                                    10.sup.3                           ______________________________________                                        at t = 0.5 h                                                                           +++     +++     +++   ++    +     +/-                                  at t = 2 h +++ +++ +++ +++ +++ +++                                          ______________________________________                                    

EXAMPLE 13

Complementary tests were performed on batches of urine taken from 50people (35 people suffering from a bacterial urinary infection and 15people, used as controls, not suffering from a urinary infection). Thegelled systems of Examples 1-5 were used simultaneously for each urinesample tested. 2 ml of urine were introduced into each of the centrifugetubes.

After centrifugation at 3000-5000 g for 15 minutes at RT, followed byculture in situ at 37° C. for 24 h, the Sen, Spe and NPV (for lowbacterial concentrations) observed with these urine samples wereimproved when using the gelled systems according to the invention,compared with the above-mentioned Bac-T-Screen® and FiltraCheck®-UTItests. Some of the results obtained are collated below for aconcentration of 10³ bacteria/ml in the urine samples:

Sen=95-98%

Spe=93-97%

NPV=91-94%

EXAMPLE 14

The gelled system of Example 2 was used to assess the presence orabsence of bacteria in 8 operating theater suites. The tubes (with avolume of 15 ml), packed with said gelled system and optionally providedwith a flange of the funnel type at their upper orifice, were placed inoperating theater suites for 24 h in the vicinity of the gas inlets, ata height of 1.50 m and at a distance of 1 m from the nearest wall.Distilled water was run over the tubes from a pipette in order to rinsethe exposed part of each tube and of each flange present. Aftercentrifugation (30 minutes at 3000 g) and culture in situ at 37° C. for24 h, it was possible to detect the presence of bacteria (mainly strainsof Staphylococcus) in the ambient atmosphere of one of the operatingtheater suites. The contaminated operating theater suite was sterilized.

EXAMPLE 15

The filters (cellulose membranes) of the ventilation ducts of 6operating theater suites were cut up and then finely dilacerated. Thematerial obtained was triturated with distilled water; the resultingsupernatant was introduced at a rate of 1 to 2 ml into tubes obtained bythe process of Examples 2 and 11. After centrifugation at 3000-5000 gfor 15 minutes at RT, followed by culture in situ at 37° C. for 24 h, itwas possible to detect the presence of bacteria (mainly strains ofPseudomonas) in one of the six operating theater suites and the presenceof yeasts (strains of Aspergillus in this instance) in another of saidoperating theater suites. The two contaminated operating theater suiteswere sterilized.

EXAMPLE 16

"Clarified" (i.e. delipidated) and sterilized milk was contaminated withstrains of Candida (at a concentration greater than or equal to 10⁵germs/ml) and distributed at a rate of 2.5 ml into batches of centrifugetubes (5 tubes per batch) prepared by the process of Example 7:

batch A containing isolated strains of Candida albicans,

batch B containing isolated strains of Candida glabrata,

batch C containing isolated strains of Candida krusei,

batch D containing non-isolated strains of various Candida (i.e. strainsof Candida albicans associated with other strains of Candida), and

the control batch consisting of said clarified and sterilized milk.

After centrifugation at 3500-4000 g for 20 minutes at RT, followed byculture in situ at 37° C. for 1-2 h, it was found that the gelled systemof Example 7 gave a positive color reaction with the tubes of batches Aand D and did not give a positive color reaction with batches B and Cand the control batch.

EXAMPLE 17

A hemoculture sample (taken from an infected patient) which has beenbrought into contact beforehand with saponin (to lyze the erythrocytespresent) is introduced at a rate of 1 to 3 ml into a centrifuge tubepacked with the gelled system prepared by the process of Example 4. Thetube is centrifuged at 3000-5000 g for 15 minutes at RT. After culturein situ at 37° C. for 1-2 h, a positive color reaction is observed insaid centrifuge tube, indicating the presence of Gram-negative bacteriain the hemoculture.

In a modified embodiment, by introducing an antibiotic into thedevelopment phase or into the combined development phase/absorptionphase so as to have a set of several tubes each containing a differentantibiotic, it is possible to assess the resistance of the bacteriacontained in hemocultures towards customary antibiotics.

EXAMPLE 18

Some beef is cut up and finely dilacerated. Isotonic solution andbacteria (10⁷ germs/ml of strains of Staphylococcus epidermidis) areadded to the resulting material. The supernatant is collected andintroduced at a rate of 1-2 ml into a tube containing the gelled systemprepared by the process of Example 2.

The tube is centrifuged at 3000-5000 g for 15 minutes at RT culture insitu at 37° C. for 24 h. A color change is observed in the lower part ofthe development phase, confirming the presence of bacteria in the sampletested.

EXAMPLE 19

The following are introduced successively into a transparent plasticcentrifuge tube with a height of 30 mm, which is provided with acapillary (Φ=2.2 mm; volume=0.045 ml) and corresponds to FIG. 10, thetube being centrifuged after each introduction (1000-3000 g for 10-20minutes at RT) in order to compress each layer:

0.045 ml of a gel of G 100/aqueous culture medium, in which the G 100has a concentration by dry weight of 0.2 g/ml and a particle size in theswollen state of 103-311 μm, this medium containing resazurin, and

0.5 ml of paraffin oil.

A urine sample from a patient suffering from a urinary infection causedby strains of Escherichia coli is then introduced. After 20 minutes at3000-5000 g at RT, the coloration of the characteristic of the presenceof bacteria is observed 1 hour after centrifugation was begun.

The bacteria are counted optically (spectrometer, densitometer orMacFARLAND apparatus for measurement of the turbidimetry), ifappropriate with standard urine solutions each containing a known amountof Escherichia coli.

The development phase is recovered, as explained above, by means of asecond centrifuge tube containing the aqueous recovery solution. This isused to establish antibiograms for assessing the resistance of thebacteria collected towards several antibiotics.

Particularly effective results are obtained when the volume of thecapillary 7 is reduced to 0.02-0.03 μl.

According to the invention, an assay kit is recommended which comprisesat least

(1) a conical-bottomed centrifuge tube 6a containing a gelled systemconsisting, from top to bottom, of the protection phase 4, theabsorption phase 2 and the development phase 1, a spherical-bottomedcentrifuge tube 6b containing a gelled system consisting, from top tobottom, of the protection phase 4, the absorption phase 2, thedevelopment phase and the barrier phase 3, or a capillary-bottomedcentrifuge tube 6c containing a gelled system consisting of thedevelopment phase on which the protection layer and/or the combinedprotection layer+absorption layer rest, if appropriate; and/or

(2) bottles containing the constituent materials (i.e. the gel or gels,the particulate substance, the solid paraffin and/or the liquidparaffin) of said phases, the reagent which induces a detectablevariation in optical measurement in the presence of microorganisms and,if appropriate, the culture medium for the swelling of said developmentphase 1.

It is also recommended to use the method and the gelled system accordingto the invention for

detecting the presence or absence of bacteria in a blood or urinesample, especially in the field of hemoculture screening and urinescreening,

identifying a strain of microorganism in a biological sample by using,in the development phase, (α) a reagent which induces a detectablevariation in optical measurement, and (β) a culture medium, both ofwhich are specific for said strain,

counting the microorganisms present in the development phase,

collecting the development phase containing microorganisms for thepurpose of studying the latter, or

determining the resistance of strains of microorganisms towards a groupof customary antibiotics.

What is claimed is:
 1. A centrifuge tube comprising a cylinder portion;a frustoconical portion; a capillary at a bottom of the tube; andcontaining a gel system for detecting microorganisms, said gel systemcomprising:a development phase comprising a gel containing microorganismculture medium and a reagent capable of inducing a detectable variationin optical measurement in the presence of microorganisms and a firstphase intimate mixture which comprises water originating wholly orpartly from the culture medium and water-absorbing polymer particles sothat, in said first phase intimate mixture, said polymer particles havea) a concentration by dry weight of between 0.05 g/ml and 0.2 g/ml andb) a diameter in a swollen state of between 90 μm and 320 μm.
 2. Thecentrifuge tube of claim 1, wherein an angle α of the frustoconicalportion is less than 30° and the development phase is found in thecapillary.
 3. The centrifuge tube of claim 1, wherein the microorganismscan be counted.
 4. The centrifuge tube of claim 1, wherein themicroorganisms can be recovered from the development phase.
 5. Thecentrifuge tube of claim 1, further comprising:a protection phase whichis an inert oil with a density less than that of water and wherein saidprotective phase rests o n said development phase.
 6. The centrifugetube of claim 5, wherein said inert oil is paraffin oil.
 7. Thecentrifuge tube of claim 1, further comprising:an absorption phaseessentially impermeable to water and substances dissolved therein, saidabsorption phase during centrifugation retaining the insoluble particlescontained in said liquid sample which have a size less than 5 μm butletting through particles with a size greater than or equal to 5 μm,said absorption phase comprising (i) a gel or (ii) silica or silicateparticles with a size less than 0.1 μm, said absorption phase rests onsaid development phase and said protection phase rests on saidabsorption phase.
 8. The centrifuge tube of claim 7, wherein saidabsorption phase is a gel consisting of a second phase intimate mixtureof water and water-absorbing polymer particles so that, in said secondphase intimate mixture, said polymer particles have a) a concentrationby dry weight to water of between 0.2 g/ml and 1 g/ml and b) a diameterin a swollen state of between 160 μm and 530 μm.
 9. The centrifuge tubeof claim 7, wherein said absorption phase is a layer consisting of amineral substance which is micronized so as to have a particle size lessthan 0.1 μm in the dry state and does not essentially swell in thepresence of water.
 10. An assay kit for detecting microorganisms,comprising:a conical-bottomed centrifuge tube containing a gel system,said gel system comprising:a development phase comprising a gelcontaining microorganism culture medium and a reagent capable ofinducing a detectable variation in optical measurement in the presenceof microorganisms and a first phase intimate mixture which compriseswater originating wholly or partly from the culture medium andwater-absorbing polymer particles so that, in said first phase intimatemixture, said polymer particles have a) a concentration by dry weight ofbetween 0.05 g/ml and 0.2 g/ml and b) a diameter in a swollen state ofbetween 90 μm and 320 μm; a protection phase which is an inert oil witha density less than that of water; and an absorption phase essentiallyimpermeable to water and substances dissolved therein, said absorptionphase during centrifugation retaining the insoluble particles containedin said liquid sample which have a size less than 5 μm but lettingthrough particles with a size greater than or equal to 5 μm, saidabsorption phase comprising (i) a gel or (ii) silica or silicateparticles with a size less than 0.1 μm, wherein said absorption phaserests on said development phase and said protection phase rests on saidabsorption phase.
 11. An assay kit for detecting microorganismscomprising:a spherical-bottom centrifuge tube containing a gel system,said gel system comprising:a development phase comprising a gelcontaining microorganism culture medium and a reagent capable ofinducing a detectable variation in optical measurement in the presenceof microorganisms and a first phase intimate mixture which compriseswater originating wholly or partly from the culture medium andwater-absorbing polymer particles so that, in said first phase intimatemixture, said polymer particles have a) a concentration by dry weight ofbetween 0.05 g/ml and 0.2 g/ml and b) a diameter in a swollen state ofbetween 90 μm and 320 μm; a protection phase which is an inert oil witha density less than that of water; an absorption phase essentiallyimpermeable to water and substances dissolved therein, said absorptionphase during centrifugation retaining the insoluble particles containedin said liquid sample which have a size less than 5 μm but lettingthrough particles with a size greater than or equal to 5 μm, saidabsorption phase comprising (i) a gel or (ii) silica or silicateparticles with a size less than 0.1 μm; and a barrier phase which cannotbe crossed after centrifugation by any microorganisms present, whereinsaid development phase rests on said barrier phase, said absorptionphase rests on said development phase and said protection phase rests onsaid absorption phase.
 12. An assay kit for detecting microorganisms,comprising:a capillary-bottomed centrifuge tube containing a gel system,said gel system comprising:a development phase comprising a gelcontaining microorganism culture medium and a reagent capable ofinducing a detectable variation in optical measurement in the presenceof microorganisms and a first phase intimate mixture which compriseswater originating wholly or partly from the culture medium andwater-absorbing polymer particles so that, in said first phase intimatemixture, said polymer particles have a) a concentration by dry weight ofbetween 0.05 g/ml and 0.2 g/ml and b) a diameter in a swollen state ofbetween 90 μm and 320 μm; and a protection phase which is an inert oilwith a density less than that of water, wherein said development phaseis found within the capillary and said protection phase rests on saiddevelopment phase.
 13. The assay kit of claim 12, further comprising:anabsorption phase essentially impermeable to water and substancesdissolved therein, said absorption phase during centrifugation retainingthe insoluble particles contained in said liquid sample which have asize less than 5 μm but letting through particles with a size greaterthan or equal to 5 μm, said absorption phase comprising (i) a gel or(ii) silica or silicate particles with a size less than 0.1 μm, whereinsaid absorption phase rests on said development phase and saidprotection phase rests on said absorption phase.
 14. The assay kit ofclaim 10, further comprising bottles containing the constituentmaterials of said phases.
 15. The assay kit of claim 10, furthercomprising a reagent which induces a detectable variation in opticalmeasurement in the presence of microorganisms.
 16. The assay kit ofclaim 10, further comprising a culture medium for the swelling of saiddevelopment phase.
 17. The assay kit of claim 11, further comprisingbottles containing the constituent materials of said phases.
 18. Theassay kit of claim 11, further comprising a reagent which induces adetectable variation in optical measurement in the presence ofmicroorganisms.
 19. The assay kit of claim 11, further comprising aculture medium for the swelling of said development phase.
 20. The assaykit of claim 12, further comprising bottles containing the constituentmaterials of said phases.
 21. The assay kit of claim 12, furthercomprising a reagent which induces a detectable variation in opticalmeasurement in the presence of microorganisms.
 22. The assay kit ofclaim 12, further comprising a culture medium for the swelling of saiddevelopment phase.